Rumen protected essential amino acids

ABSTRACT

Use of essential amino acid imines and compositions containing them as a source of rumen protected essential amino acids for ruminant animals. Preferred are histidine and methionine.

FIELD OF THE INVENTION

This invention relates to a rumen stable bioavailable dietary supplementfor ruminant animals to provide in bioavailable form essential aminoacids, preferably histidine and methionine. It represents a specificimprovement over the bypass rumen product of prior U.S. Pat. No.5,885,610 to Anderson, owned by the common Assignee.

BACKGROUND OF THE INVENTION

It is well known that ruminants are in need of bioavailable essentialamino acids in order to perform well as domesticated livestock. In thisregard, if the animal, for example a dairy cow, does not have itsminimum requirements of essential amino acids such as lysine andmethionine, the animal will not produce milk at optimum yield, and itshealth may be in general decline.

Providing essential amino acids to ruminants is not as simple as itsounds. For example, the bacteria in the rumen of a cow are known toroutinely degrade amino acid sources, like lysine and methionine. Putanother way, the bacteria in the rumen metabolize the amino acid sourceand thus “rob” the animal of the benefit of the amino acid. By the timemetabolized byproduct passes from the rumen into the intestine, theamino acid is gone. The challenge, therefore, is to develop productswhich will allow the amino acid to be stable in the rumen, but capableof absorption when it passes from the rumen into the intestine. In otherwords, the essential amino acids, such as lysine and methionine, need tobe bioavailable only in the intestine, and remain stable and thereforenot metabolized in the rumen.

In the past, this problem has been recognized, and feed developers haveused fats, minerals, carbohydrates and binders to protect amino acidsfrom rumen degradation. This technology involves simple coating of thematerial in hopes that the coated amino acid is rumen stable. Recently,Rhone Poulenc has provided a pH-sensitive polymer coating. The theory ofa pH-sensitive polymer coating for the amino acid revolves around the pHdifferential between the rumen and the intestine. The rumen, forexample, typically has a pH of 5.5 to 7.0, and the intestine a pH of2-3. The theory of polymer-coated essential amino acids is thatsomething which is stable as a coating at 5.5 to 7.0 (the rumen pH), butwill solubilize at more acid pH's of the intestine (pH 2-3), should bestable in the rumen, but available in the intestine.

Both technologies used in the past, i.e. coatings, such as fat coatings,and the more recently developed pH-sensitive polymer coatings, have metwith limited success and have some problems. The primary problem withany product relying upon coatings of any kind for rumen stability isthat the coating can become abraded during handling and during chewingby the animal. If the process handler disturbs the coating, then theamino acid becomes available to microbes in the rumen and consumed, andtherefore wasted by the animal. Likewise, if the animal abrades thecoating during chewing, it then becomes available in the rumen for rumenbacteria to metabolize, and is therefore also wasted to the animal.Additionally, fat-protected or coated essential amino acids rely uponthe fat resistance to enzymes in the rumen that are capable of digestingthe protective fat coat, and, on the other hand, the ability ofdigestion by enzymes post-rumenally. However, if there is not a properbalance between resistance to attack in the rumen and digestion in theintestine, then the amino acid benefit to the animal may be lost.

From the above description, it can be seen that there is a real andcontinuing need for the development of products for delivery ofessential amino acids to ruminant animals in a form that allows thematerial to be rumen stable, i.e. resistant to degradation in the rumen,but yet after delivery from the rumen to the intestine, highlyabsorbable and bioavailable in the intestine. It is a primary objectiveof this invention to improve upon available products to fulfill thisneed safely, effectively, efficiently and at low cost.

In the prior Zinpro Corporation patent by Michael Anderson, it wasdiscovered that calcium or magnesium complexed salts of certain aminoacids could be used to prepare a bypass rumen product. This inventiontakes a different attack on the problem with specificity of improvingthe availability of essential amino acids in ruminants.

Lysine is an essential amino acid in the diet of mammals. That is,lysine cannot be synthesized by mammals at a rate adequate to meetmetabolic requirements and so must be supplied in the diet. Corn (Zeamays L.) is notoriously low in lysine and, if used in a single grainration, requires lysine supplementation both to maintain animal healthand to achieve economical animal growth. Protected lysine iminederivatives are disclosed in copending, concurrently filed, commonlyassigned case, Stark et al. entitled RUMEN PROTECTED LYSINE.

The present invention extends the above referenced technology to otherlimiting and/or essential amino acids, by forming compounds which areessentially immune to attack by the microbes in the rumen but can stillbe digested and absorbed through the intestine wall to allow a highlybioavailable form of essential amino acids that are surprisingly immunefrom rumen organism attack. Structures of the compounds prepared arecentered around the imine of the essential amino acids (Schiff's base).

In the past, there have been some alpha imine and epsilon iminederivatives of lysine investigated for biologically available activederivatives for rats. See for example, Finot, N-Substituted Lysines AsSources of Lysine in Nutrition, Adv. Exp. Med. Bio. 1978; 105:549-570;Nutritional Improvement of Food and Feed Proteins, edited by Friedman,published at Plenum, New York, and Finot et al., Availability of thetrue Schiff's bases of lysine. Chemical Evaluation of the Schiff's BaseBetween Lysine and Lactose in Milk, Adv. Exp. Med. Biol. 1977;86B:343-365. The first Finot article concludes that the biologicalavailability of derivatives were four to seven times less reactive thanfree lysine in the Maillard reaction and could therefore be subjected toheat. The second article deals with chemical evaluation of the Schiff'sbase between lysine and the lactose in milk. There is no teaching ineither article of any compounds having usefulness of providing stabilityof lysine derivatives in the rumen or providing compounds which can besuccessfully absorbed through the intestine after passing through therumen to assure that lysine will be available to the animal for dietsupplementation of this important essential amino acid. There is also noteaching relating to other essential amino acids beyond lysine.

It is therefore another primary objective to provide diet supplements ofruminants to provide essential amino acid supplementation for animalsthat often use as a major grain ration corn (known to be notoriously lowin lysine and methionine). As a result, overall economic growth of theanimal can be enhanced, and enhanced in a manner which assures that theexpense of essential amino acid supplementation will go to the animaland not be “robbed” (so to speak) by the microbes in the rumen as thematerial passes through the rumen.

The method of achieving the above objectives with certain chemicalstructures premised around imine (Schiff's base) of the essential aminoacids is another primary objective of the invention.

Yet another objective of the present invention is to provide compoundswhich can be utilized to achieve successfully a rumen bypass anessential amino acid supplement that does not rely upon encapsulationand one which employs compounds that are easily processible in feedforms.

A still further objective is to provide methionine and histidine andother essential amino acid supplements that are rumen protected.

BRIEF SUMMARY OF THE INVENTION

Use of essential amino acid imines and compositions containing them as asource of rumen protected essential amino acids for ruminant animals.Especially preferred embodiments are methionine and histidine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The basic concept in preferred form relates to the imine (Schiff's base)of essential amino acids and some of their derivatives and salts. Itcould be the imine variety produced from aldehydes or ketones, and anyone of the essential amino acids. It could be the imines produced fromaldehydes or ketones and the amino acid methionine or histidine. Itcould also be the imines from aldehydes or ketones and salts, ester orcomplexes of these essential amino acids. In other words, the inventionrecognizes the imines of the alpha amino group as rumen resistantsources of the essential amino acids, and modification of otherfunctional groups can be made, if one wishes, or not made if one wishes.

In the ruminant animal, ingested feed first passes into the rumen whereit is partially broken down by bacterial fermentation. During rumenfermentation, rumen microbes utilize nitrogen from nitrogen compoundsthat they have degraded to form microbial protein. Nitrogen sources forrumen microbes include rumen degradable protein and peptides, free aminoacids and urea. Microbial protein and undegraded feed protein pass tothe abomasum and small intestine where hydrochloric acid and mammalianenzymes degrade microbial protein and undegraded feed protein to freeamino acids and short peptides. The amino acids and short peptides areabsorbed in the intestine, and the ruminant animals utilize the aminoacids for synthesis of protein to sustain life, grow, reproduce andproduce milk. However, if the amino acid, such as lysine or methionineor histidine, has been metabolized by rumen microbes, its value to thehost animal is lost.

Of the twenty or more amino acids utilized by the animal to synthesizeproteins, nine are considered to be essential. Examples of the essentialamino acids include leucine, isoleucine, valine, methionine, threonine,lysine, histidine, phenylalanine and tryptophan. Essential amino acidsare those amino acids which are required in quantities exceeding amountsproduced by the animal, and must be supplied by microbial protein orrumen undegraded protein. Amino acids supplied in excess are degraded bythe animal and excreted in the form of urea. The process of synthesizingurea from ammonia is a process requiring energy input from the animal.If certain essential amino acids are not provided in adequate amounts,the animal will be limited on the amount and types of protein it canproduce, thus limiting animal performance. Supplying the proper amountsof essential amino acids therefore maximizes animal performance whileenhancing efficiency of energy utilization by the animal.

Lysine and methionine are two of the most limiting essential amino acidswhen corn-based rations are fed. Results from studies also indicate thatmilk protein content is the most sensitive of the production variables(yield of milk, fat-corrected milk, milk protein, milk fat, and contentof milk fat and protein) to alterations in amino acid content ofduodenal digesta. Researchers have determined, by infusing incrementalamounts of the limiting amino acids into the duodenum of lactating dairycows, that the required contribution of lysine and methionine to totalessential amino acids in duodenal digesta for maximum milk proteincontent approximated 15% and 5.2%, respectively.

This present invention relates to essential amino acid imines as themost preferred, including some of the essential amino acids such asmethionine and histidine. While this case preferably deals withmethionine and histidine and their derivatives and salts, other alphaamino acids containing an alpha amino group may also be employed.

Compositions of the present invention which are rumen stable butintestine soluble for supplementing the diet of ruminants with a sourceof rumen protected essential amino acids can generally be described asdietary supplement compositions that are imines (Schiff's base) of thealpha amine moiety. They can be an imine from a variety of aldehydes orketones as evidenced by the examples below. The amino acid portion ofthe molecule could also be salts or esters or amides of the carboxylategroup. Generally speaking, the composition will contain a dietsupplementing effective amount of an imine of the formula:

W represents the remaining portion of an essential amino acid and can beselected from the group derived from leucine, isoleucine, valine,methionine, threonine, lysine, histidine, phenylalanine and trytophan.It preferably is from histidine or methionine in which case W is:

It goes without saying that the carboxylic acid moiety can be the aciditself or functional substituted derivatives such as salts, amides,esters, etc.

In the formula, the R₁ and R₂ moiety can be the same or different andcan be selected from the group consisting of hydrogen, aliphatics,aromatics, and cyclic moieties. R₃ can be —OH, or as previouslyindicated an ester producing moiety or a salt producing moiety or anamide producing moiety or a complex producing moiety, etc. The preferredstructures are those wherein R₁ is hydrogen and R₂ is an aromatic, andR₃ is —OH. As R₃—OH is preferred, but it is mentioned herein that thescope of the invention includes other moieties as listed, primarily sothat someone simply cannot substitute essentially any other moiety andstill achieve the benefit of the invention and argue non-infringement.

The preferred moieties at the R₁ and/or R₂ position are those formedfrom use as a reactant Benzaldehyde, Salicylaldehyde, cinnamaldehyde, orvanillin to prepare the most preferred compounds falling within thegeneric formula of structure 1 as herein set forth. These are shownbelow.

The compounds prepared above and especially those listed as preferredare easily processable. They can be sold and subdivided as asupplementing additive or they can be mixed with carriers to improvepackaging, processability, and taste. Preferred carriers are, forexample, powdered sugar which significantly improves taste for theruminants that ingest the same. For example, the Benzaldehydederivatives have an almond taste which can be masked with powderedsugar.

While it is preferred that the compounds of the present invention beadded without additional carriers or filler material, as heretoforementioned flavorants can be used as or with the carrier. If carriers areemployed, the carrier can be suitable carriers such as distillersfermentation solubles, feed grains, corn cob flour, whey, or othercellulosic carrier materials. They can also be added at the same time astrace mineral preparations are added. In other words, they can be mixedwith other nutritional ingredients.

The amount of supplement added to the feed ration will vary, of course,depending on whether one is using the pure compositions or thecomposition with a carrier. Basically the supplement will simply mixwith the feed ration, as sold.

Generally the imines should be added at a level to provide sufficientessential amino acid for the animals daily nutritional needs, i.e.,within the range of about 1 gram to about 50 grams per animal per day.

The following examples illustrate the preparation of the imine (Schiff'sbase) of lysine, methionine and histidine of the present invention andillustrate a variety of different moieties that may be attached at theR₁ through R₃ position.

EXAMPLE 1 Preparation of N-Benzylidene-L-Lysine from lysinehydrochloride and benzaldehyde

Lysine hydrochloride (4.8 gr, 26.3 mmol) was dissolved in 35 mL ofwater. NaOH (1 gr, 26.3 mmol) was added to the mixture which was cooledwith an ice batch. To this mixture was added benzaldehyde (2.8 gr, 26.3mmol) and the product precipitated in about 10 minutes. The mixture wasfiltered and washed with water. The solid was dried to yield about 5.2gr of a white solid.

EXAMPLE 2 Preparation of 2-amino-6-((E)-3-phenylallylideneamino)hexanoicfrom lysine hydrochloride and trans cinnamaldehyde

Lysine hydrochloride (4.2 gr, 23 mmol) was dissolved in 30 mL of water.NaOH (0.91 gr, 23 mmol) was added to the mixture which was cooled withan ice batch. To this mixture was added trans cinnamaldehyde (3.0 gr 23mmol) and the product precipitated in about 10 minutes. The mixture wasfiltered and washed with water. The solid was dried to yield about 4.9gr.

EXAMPLE 3 Preparation of 2-amino-6-(4-methoxybenzylideneamino)hexanoicacid from lysine hydrochloride and 4-hydroxy-3-methoxybenzaldehyde

Lysine hydrochloride (2.2 gr, 12 mmol) was dissolved in 25 mL of water.NaOH (0.48 gr, 12 mmol) was added to the mixture which was cooled withan ice batch. To this mixture was added 4-methoxybenzaldehyde (1.6 gr,12 mmol) and the product precipitated in about 10 minutes. The mixturewas filtered and washed with water. The solid was dried to yield about2.6 gr.

EXAMPLE 4 Preparation of 2-amino-6-(octylideneamino)hexanoic acid fromlysine hydrochloride and octylaldehyde

Lysine hydrochloride (2.7 gr, 14.8 mmol) was dissolved in 100 mL ofwater. NaOH (0.59 gr, 14.8 mmol) was added to the mixture which wascooled with an ice batch. The pH was adjusted to between 4-5 by theaddition of acetic acid. To this mixture was added octylaldehyde (1.9gr, 14.8 mmol) and the product precipitated in about 10 minutes. Theprecipitate agglomerated as an oil. The solvent was decanted off and theoil residue dried under vacuum. This yielded 2.5 gr as an oil.

EXAMPLE 5 Evaluation of Feeding N-Benzylidene-L-Lysine Using LactatingHolstein Dairy Cows

-   TREATMENT DURATION: 14 days-   TREATMENTS: 1) Control    -   2) Control plus 40 g Lys from N-Benzylidene-L-Lysine

All cows received the same base diet. Cows were assigned to one of twotreatments: (1) control, (2) control+N-Benzylidene-L-Lysine (TrTA).N-Benzylidene-L-Lysine was supplemented at a rate to supply 40 g of Lyswhen cows consumed 53.0 lb dry matter/d. The control diet was formulatedto be deficient of lysine. The lactation response to the control diet aswell as the diet supplemented with N-benzylidene-L-lysine is shown inTable 1. “P” refers to the probability value (p-value).

TABLE 1 Lactation responses to N-Benzylidene-L-Lysine Item Control Trt AP Milk, lbs/d 73.7 77.3 .1743 FCM, lbs/d 76.0 81.2 .1074 ECM, lbs/d 75.981.3 .0698 Fat, % 3.71 3.81 .5358 Fat, lbs/d 2.72 2.94 .1420 Protein, %3.06 3.14 .1478 Protein, lbs/d 2.25 2.41 .0344 SCC 230.9 239.3 .5014DMI, lbs/d 53.5 53.1 .8487 ECM/Feed 1.45 1.53 .2562

From the Table 1 data, it can be concluded that the imine supplementcomposition produced more milk, more protein and more fat, and that thedifferences were significant as indicated by the P value. In contrast,if one were to simply add lysine to the diet, one would see nodifferences such as these, since microbes in the rumen would simplyconsume the lysine never allowing it to effectively supplement theanimal and produce any different or enhanced result.

It therefore can be seen that the invention accomplishes at least all ofits stated objectives.

EXAMPLE 6 Preparation of sodium2-(benzylideneamino)-4-(methylthio)butanoate from methionine andbenzaldehyde

Methionine (2.2 gr, 14.7 mmol) was added to 100 mL of MeOH. NaOH (0.59gr, 14.7 mmol) was added to the mixture and stirred until all componentsdissolved. To this mixture was added benzaldehyde (1.9 gr, 17.9 mmol)and the mixture was stirred for about 10 minutes. The mixture was thenconcentrated under vacuum and the residue was added to EtOH. The productcrystallized from this solution. It was filtered and washed with EtOH.The solid was dried to yield about 1.5 gr. The product was white solidand analyzed for percent nitrogen and compared to theoretical.Theoretical 5.9% nitrogen, actual 5.9%.

EXAMPLE 7 Preparation of sodium2-(benzylideneamino)-3-(1H-imidazol-4-yl)propanoic acid from histidinehydrochloride and benzaldehyde

Histidine monohydrochloride monohydrate (5.3 gr, 25.3 mmol) was added to100 mL of MeOH. NaOH (2.0 gr, 50 mmol) was added to the mixture andstirred The histidine did not completely dissolve so water was addeduntil all components dissolved. To this mixture was added benaldehyde(2.6 gr, 25 mmol) and the mixture was stirred for about 10 minutes. Themixture was then concentrated under vacuum and the residue was added toEtOH. The product was filtered and washed with EtOH. The solid was driedto yield about 3.7 gr and was a white solid.

1. A composition for supplementing the diet of ruminants with a source of rumen protected essential amino acid comprising: a non toxic carrier; and a diet supplementing effective amount of an imine derivative of an essential amino acid of the formula:

wherein R₁ and R₂ can be the same or different and are selected from the group consisting of hydrogen, aliphatic, aromatics, and cyclic moieties, W represents the remaining portion of an essential amino acid and R₃ can be —OH, an ester producing moiety, or an inorganic salt producing moiety or amide producing moiety.
 2. The composition of claim 1 wherein W is an organic moiety of methionine or histidine.
 3. The composition of claim 1 wherein R₁ is hydrogen and R₂ is formed from use as a reactant Benzaldehyde, Salicylaldehyde, Cinnamaldehyde or Vanillin and R₃ is —OH.
 4. The composition of claim 1 wherein R₁ is hydrogen and R₂ is formed from use as a reactant butyrlaldehyde, isobutylylaldehyde, propionaldehyde or octylaldehyde and R₃ is —OH.
 5. The composition of claim 1 wherein R₁ and R₂ are the same and are formed from use as a reactant Cyclohexanone, propiophenone, diethylketone, octanone, decanone or acetone.
 6. The composition of claim 1 wherein R₃ is either an organic moiety to form an ester or an inorganic salt moiety.
 7. The composition of claim 1 wherein the non-toxic carrier is a flavorant.
 8. The composition of claim 1 wherein the nontoxic carrier is selected from the group consisting of sugars, fermentations solubles, feed grains, corn cob flour, whey, and other cellulosic carrier materials.
 9. The composition of claim 1 wherein the diet supplementing effective amount is an amount sufficient to provide a level of from about 1 gr to about 50 gr of amino acid used per animal per day.
 10. The method of providing rumen protected lysine for dietary supplementation of ruminants, said method comprising: selecting at least one essential amino acid imine to be used as a source of rumen protected essential amino acid.
 11. The method of claim 10 wherein the amino acid imine is at least one amino acid imine of the formula:

wherein R₁ and R₂ can be the same or different and are selected from the group consisting of hydrogen, aliphatic, aromatics, and cyclic moieties, W represents the remaining portion of an essential amino acid, and R₃ can be the same or different and are selected from the group consisting of hydrogen, aliphatic, aromatic and carbonyl moieties, and where R₃ is carbonyl, R₄ is not present and R3 can be —OH, an ester producing moiety, or an inorganic salt moiety or an amide moiety.
 12. The method of claim 10 wherein W is either a histidine or methionine moiety.
 13. The method of claim 11 wherein R₁ is hydrogen and R₂ is formed from use as a reactant Benzaldehyde, Salicylaldehyde, Cinnamaldehyde or Vanillin and R₃ is —OH.
 14. The method of claim 11 wherein R₁ is hydrogen and R₂ is formed from use as a reactant butyrlaldehyde, isobutylylaldehyde, propionaldehyde or octylaldehyde and R₃ is —OH.
 15. The method of claim 11 wherein R₃ is either an organic moiety to form an ester or an inorganic moiety to form a salt.
 16. The method of claim 11 wherein an additional step includes mixing the essential amino acid imine with a non-toxic carrier as a flavorant.
 17. The method of claim 16 wherein the nontoxic carrier is selected from the group consisting of sugars, fermentations solubles, feed grains, corn cob flour, whey, and other cellulosic carrier materials.
 18. The method of claim 17 wherein a diet supplementing effective amount of said lysine imine is used and is an amount sufficient to provide a level of from about 1 gr to about 50 gr of amino acid used per animal per day. 